Optimal Seeding Densities for In Vitro Chondrogenesis of Two- and Three-Dimensional-Isolated and -Expanded Bone Marrow-Derived Mesenchymal Stromal Stem Cells Within a Porous Collagen Scaffold

摘要

Bone marrow-derived mesenchymal stromal stem cells (BMSCs) are a promising cell source for treating articular cartilage defects. The objective of this study was to assess the impact of cell seeding density within a collagen I scaffold on in vitro BMSC chondrogenesis following isolation and expansion in two-dimensional (2D) and three-dimensional (3D) environments. It was hypothesized that both expansion protocols would produce BMSCs capable of hyaline-like chondrogenesis with an optimal seeding density of 10 × 10⁶ cells/cm³. Ovine BMSCs were isolated in a 2D environment by plastic adherence, expanded to passage two in flasks containing an expansion medium, and seeded within collagen I scaffolds at densities of 50, 10, 5, 1, and 0.5 × 10⁶ BMSCs/cm³. For 3D isolation and expansion, aspirates containing known quantities of mononucleated cells (bone marrow-derived mononucleated cells [BMNCs]) were seeded on scaffolds at 50, 10, 5, 1, and 0.5 × 10⁶ BMNCs/cm³ and cultured in the expansion medium for an equivalent duration to 2D expansion. Constructs were differentiated in vitro in the chondrogenic medium for 21 days and assessed with reverse-transcription quantitative polymerase chain reaction, safranin O staining, histological scoring using the Bern Score, collagen immunofluorescence, and glycosaminoglycan (GAG) quantification. Two-dimensional-expanded BMSCs seeded at all densities were capable of proteoglycan production and displayed increased expressions of aggrecan and collagen II messenger RNA (mRNA) relative to pre-differentiation controls. Collagen II deposition was apparent in scaffolds seeded at 0.5–10 × 10⁶ BMSCs/cm³. Chondrogenesis of 2D-expanded BMSCs was most pronounced in scaffolds seeded at 5–10 × 10⁶ BMSCs/cm³ based on aggrecan and collagen II mRNA, safranin O staining, Bern Score, total GAG, and GAG/deoxyribonucleic acid (DNA). For 3D-expanded BMSC-seeded scaffolds, increased aggrecan and collagen II mRNA expressions relative to controls were noted with all densities. Proteoglycan deposition was present in scaffolds seeded at 0.5–50 × 10⁶ BMNCs/cm³, while collagen II deposition occurred in scaffolds seeded at 10–50 × 10⁶ BMNCs/cm³. The highest levels of aggrecan and collagen II mRNA, Bern Score, total GAG, and GAG/DNA occurred with seeding at 50 × 10⁶ BMNCs/cm³. Within a collagen I scaffold, 2D- and 3D-expanded BMSCs are capable of hyaline-like chondrogenesis with optimal cell seeding densities of 5–10 × 10⁶ BMSCs/cm³ and 50 × 10⁶ BMNCs/cm³, respectively.